Shanxi aged vinegar (SAV), as a natural functional ingredient, exhibits high antioxidant activities. This study aimed to investigate the protective effects of SAV on ethanol-induced liver injury and elucidate the underlying molecular mechanisms. The results showed that SAV attenuated ethanol-induced hepatotoxicity by restoring cell viability in LO2 cells, relieving the pathological changes in mice livers and reducing elevated liver enzymes. In addition, SAV (2.500 mL/kg b.w.) significantly decreased ethanol-induced reactive oxygen species (ROS) generation, which subsequently inhibited cell apoptosis and malonaldehyde (MDA) levels and reversed depletion of antioxidative defense system in mice livers. Furthermore, SAV ameliorated ethanol-induced oxidative stress and inflammation by down-regulating the expression of cytochrome P450 2E1 (CYP2E1) enzyme and NADPH oxidase. These data suggest that SAV, a health-promoting antioxidant food, exhibits hepatoprotective effects. The hepatoprotective mechanisms of SAV were associated with alleviation of ethanol-induced oxidative stress through inhibiting CYP2E1 enzyme and NADPH oxidase activities.